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Method for quantitatively detecting Vibrio parahemolyticus viable cell bacteria in food by combining PMA with microdrop digital PCR

A technology for quantitative detection of hemolytic Vibrio, which is applied in the field of quantitative detection of live bacteria of Vibrio parahaemolyticus in food, which can solve the problems of inability to accurately quantify pathogenic live cells, inability to distinguish dead and live bacteria, and interference with the accuracy of test results, etc. , to reduce the influence of amplification reaction inhibitors, avoid false negative results, and achieve high accuracy

Inactive Publication Date: 2018-12-18
INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, none of these methods can distinguish between live and dead bacteria, and the high false positive results also greatly interfere with the accuracy of the test results, making it impossible to accurately quantify the pathogenic live cells in the sample.

Method used

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  • Method for quantitatively detecting Vibrio parahemolyticus viable cell bacteria in food by combining PMA with microdrop digital PCR
  • Method for quantitatively detecting Vibrio parahemolyticus viable cell bacteria in food by combining PMA with microdrop digital PCR
  • Method for quantitatively detecting Vibrio parahemolyticus viable cell bacteria in food by combining PMA with microdrop digital PCR

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Embodiment 1: Primer specificity experiment result

[0042]Strain information for specific experiments: non-related species strains: Salmonella (ATCC 14028), Enterobacter sakazakii (ATCC12868), Escherichia coli (ATCC 25928), Staphylococcus aureus (CMCC 26001); related species strains: parahaemolyticus Vibrio alginolyticus (ATCC 17802), Vibrio alginolyticus (CMCC 1833), Vibrio riverina (CMCC 1613), Vibrio vulnificus (ATCC27562), Vibrio minimal (CMCC 1969), Vibrio mimicus (ATCC33847).

[0043] After recovery and activation of the strain, inoculate it into the enrichment medium to prepare a bacterial suspension, take 1 mL of the bacterial suspension into a 1.5 mL sterilized centrifuge tube, centrifuge at 12000 r / min for 1 min, discard the supernatant, and add 200 μL of 10% Chelex-100 to the precipitate Solution, shake vigorously for 5-10s; take out the water bath at 56°C for 30 minutes, take out and shake, heat at 95°C for 10 minutes, shake vigorously for 5-10s; centrifuge...

Embodiment 2

[0050] Embodiment 2: plate count, PMA-qPCR and PMA-ddPCR take 10 to the detection of different ratio dead / living bacteria -2 The diluted bacterial suspension was heat-killed at 95°C for 10 minutes as the dead bacterial suspension, and different volumes of 10 -2 Dilute live bacteria suspensions were mixed so that the ratio of live bacteria to the total bacteria was 0%, 1%, 5%, 10%, 20%, 50%, 70%, and 100%, and the specific addition ratio was shown in Table 2 , plate-spreading and counting the mixed bacterial solution in different proportions, then add 8 μL of PMA working solution to 1 mL of the mixed bacterial solution to make the final concentration 16 μg / mL, incubate in the dark for 10 minutes, and then use HG-EMA nucleic acid light labeling The instrument was exposed for 8 minutes respectively, and the control group was not treated with PMA. DNA was extracted according to the Chelex-100 method in Example 1, and then qPCR reaction and ddPCR reaction were performed. The qPCR r...

Embodiment 3

[0057] Embodiment 3: PMA-ddPCR sensitivity test to Vibrio parahaemolyticus detection

[0058] Inoculate the strain of Vibrio parahaemolyticus into the APW medium to prepare the bacterial suspension, adjust the turbidity of the bacterial suspension to 0.8 with a bacterial turbidimeter, use the bacterial suspension at this time as the initial dilution, and perform a gradient dilution of the bacterial solution , and plate counts were performed simultaneously. Take the diluted concentration as 10 6 ~10 1 500 μL of cfu / ml bacterial suspension was treated with PMA according to Example 2, and ddPCR was carried out after extracting DNA according to the Chelex-100 method in Example 1. The reaction system and conditions were the same as in Example 2. from image 3 It can be seen that the detection sensitivity of PMA-ddPCR to Vibrio parahaemolyticus is 2×10 1 cfu / mL, the measured copy number concentration was 1.55 copies / μL. According to the requirements of "National Food Safety Sta...

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Abstract

The invention discloses a method for quantitatively detecting Vibrio parahemolyticus viable cell bacteria in a food by combining PMA with microdrop digital PCR. The method comprises the following steps: (1) collection and processing of a sample; (2) extraction of DNA from the sample to be tested; and (3) detection of the tlh gene of Vibrio parahemolyticus by adopting a ddPCR amplification primer and a probe primer. The method has the advantages of high primer specificity, high sensitivity, high accuracy, accurateness in quantification and rapidness in detection; and a molecular gene amplification technology is adopted, so traditional culture enrichment process is needed, the whole detection process is completed in only 3-4 h, a detection result is basically same to that of traditional plate counting methods, and the detection time is shortened by 3-5 d.

Description

technical field [0001] The invention belongs to the field of food microorganism detection, and in particular relates to a method for quantitatively detecting live Vibrio parahaemolyticus bacteria in food by combining PMA and microdroplet digital PCR. Background technique [0002] Vibrio parahaemolyticus (Vibrio parahaemolyticus, Vp) is a halophilic foodborne pathogenic bacteria belonging to the genus Vibrio, mainly found in seawater near the coast, seabed sediments, fish, shrimp, and shellfish and salted processed aquatic products. The detection rate of Vibrio parahaemolyticus in coastal seawater in East China is 47.5%-66.5%, and the average disease-carrying rate of marine fish and shrimp is 45.6%-48.7%, even as high as 90% in summer. It is the primary cause of bacterial food poisoning in my country. pathogenic bacteria. The traditional quantitative detection method of Vibrio parahaemolyticus is mainly the culture counting method, which detects the isolated and cultured s...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851C12Q1/06
CPCC12Q1/6851C12Q2563/159C12Q2563/107Y02A50/30
Inventor 翁文川谢会袁幕云凌莉常彦磊谢淑媚
Owner INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU